The present invention is concerned with virus production in tissue culture using a chemically defined medium. The term "chemically defined medium" is used in virology and tissue culture to refer to culture media of known chemical composition, both quantitatively and qualitatively, in contrast to natural or undefined media containing natural products such as animal serum, embryo extracts, yeast hydrolysates, etc. of unknown or incompletely known chemical constitution.
A number of chemically defined media are known. Most of these are solutions of nutrients, e.g., carbohydrates, lipids and amino acids, with vitamins, salts and minerals, and often containing other nutrient materials such as purine bases, adenosine triphosphate, etc.
The media are generally used as a solution in a balanced salt solution ("BSS"), a solution balanced in quantity and ratio of ionic species so as to be physiologically acceptable in pH, mineral content, osmotic pressure, etc. A number of balanced salt solutions are in widespread use, e.g., Hank's BSS Earle BSS; Dulbecco Phosphate Buffered Saline, Puck saline, and others. A number of chemically defined media likewise widely used, e.g., Medium 199 of Morgan, Morton and Parker, Proc. Soc. Exper. Biol. & Med. 73: 1-8 (1950); Eagle Basal Medium, Science 122:501-4 (1955); Science 123: 845-7 (1956); Eagle, et al, J. Biol. Chem. 226: 191-206 (1957); Eagle Minimum Essential Medium, Eagle, Science 130: 432-7 (1959); Trowell Medium T8, Exper. Cell Res. 16: 118-147 (1959); Waymouth MB 752/1 Medium, J. Nat. Cancer Inst. 22: 1003-17 (1959); Puck N 16 Puck, Cieciura et al. J. Exper. Med. 108: 945-956 (1958) and Neuman and Tytell, Proc. Soc. Exptl. Biol. Med., 104: 252-256 (1960). Details for preparation and formulation of such media are found in conventional reference texts, such as Handbook of Cell and Organ Culture, by Merchant, Kahn and Murphy, Burgess Publ. Co., Minneapolis (1960); and In Vitro Monograph No. 1, A Survey of Commercially Available Tissue Culture Media, by Helen C. Morton, In Vitro (J. Tissue Culture Association), Vol. 6, No. 2, pp. 89-108 (1970).
In addition to classification of media as "defined" or "undefined" media, they are also classified broadly on the basis of ability to support cell metabolism and proliferation. Media which permit metabolism of cells at levels adequate for many purposes, including virus production, but which either do not support significant cellular proliferation at all or which support only marginal limited proliferation, are referred to as "maintenance media". Media which support cellular proliferation are generally referred to as "growth media". Most growth media have as their base a BSS or a chemically defined medium which is supplemented with one or more natural products, usually animal serum. Typical growth media include either Eagle's Basal Medium or Medium 199 supplemented with 10 to 20 percent whole animal serum, BSS with 40 percent serum; and 40 percent BSS with 40 percent serum and 20 percent embryo extract. Some chemically defined protein-free media can be used as growth media for a limited number of cell lines. Katsuta and Takaoka, Methods in Cell Biology VI, Ed. by Prescott, Academic Press (1973) Chapter 1. However, for growth of primary cells in tissue culture, and for most large scale tissue culture for vaccine production, a serum supplement has been necessary.
The disadvantages of using serum in vaccine production are well known. Suitable serum is difficult and expensive to obtain, store and use; it is a source of undesirable foreign proteins which may be carried into the final vaccine product; its composition varies from lot to lot; and it is a potential source of contaminating viruses or Mycoplasma. Esber et al., J. National Cancer Institute, 50: 559-62 (1973), Barile and Kerne, Proc. Soc. Exp. Biol. Med. 138: 432-7 (1971); Merril et al., In Vitro, 8: 91-3 (1972).